MICHELSONPMORLEY PRIZE LECTURE BIOMED TARGETING OF MICROSCOPIC ULTRAFAST OPTICS

为生物研究和细胞成像提供的海报 药物开发会议(剑桥医疗技术研究所，12-14 1998年11月，Sandiego)荧光的多光子(NP)激发 是基于多个光子可以相互作用的原理 同时与荧光团结合，从而将它们的能量增加到 以每一个人数倍的能量刺激一次过渡 《光子》(Denk 1990；Williams 1994；Denk 1995；Xu 1996；Xu 1996)。 5-羟色胺吸收带在波长小于250纳米处存在；因此 2P和3P的吸收波长分别为~500和~700 nm。 在光激发后，5-羟色胺分子振动地松弛 通过环境相互作用释放出荧光光子 能量小于总吸收能量(~340 nm)。 在这些成像研究中，我们使用了低能量的红光子(740 Nm) 以及激活天然5-羟色胺的三光子机制 荧光。红光3P激发缓解了对深紫外线的需求 光学及其相关的象差和低透射率。 非线性激发也在本质上局限于焦点 平面和 因此，与单光子技术相比，这种技术具有更小的侵入性。我们 以前曾显示过5-羟色胺颗粒的3P显微镜 RBL-2H3细胞是可能的(Maiti 1997)。在这里，我们报告3P成像 对那些经历抗原刺激分泌的细胞进行测量。 每隔10-20秒采样一帧，我们观察每个水泡的 内容物突然消失，没有颗粒运动的证据。 单颗粒释放度可测定并指示释放 这在空间上是耦合的，有时是振荡的。在出走时 颗粒通常会留下清晰的脚印，这表明 未溶解的5-羟色胺仍与颗粒基质结合。丹克，W. D.W.活塞和W.W.韦伯(1995)。双光子分子激发 在激光扫描显微镜下。生物共聚焦手册 显微镜。J.B.波利。纽约，全会出版社：445-458。丹克， J.H.Strickler和W.W.Webb(1990)。“双光子激光扫描 首页--期刊主要分类--期刊细介绍--期刊题录与文摘--期刊详细文摘内容 (1997年)。用来测量活细胞中5-羟色胺的分布 《科学》275：530-532.威廉姆斯，R.M.，D.W. Piston和W.W.Webb(1994)。“双光子分子激发” 为激光显微镜提供固有的3维分辨率 和显微光化学。《FASE B J.8：804-813》。徐，C，W.W. Webb(1996)。双光子激发截面的测量 含690至1050纳米数据的分子荧光团>，J.Opt.SoC。 上午好。B 13(3)：481-491。Xu，C.，等人。(1996年)。“多光子” 荧光激子：生物新的光谱窗口 非线性显微镜>，《学报》，科学版，93：10763-10768。

Poster given for the Cellular Imaging for Biological Research and Drug Development meeting (Cambridge Healthtech Institute, 12-14 November 1998, SanDiego) Multi-photon (nP) excitation of fluorescence is based on the principle that multiple photons can interact "simultaneously" with a fluorophore and thereby add their energies to excite a transition with multiple times the energy of each individual photon (Denk 1990; Williams 1994; Denk 1995; Xu 1996; Xu 1996). Serotonin absorption bands exist at wavelengths less than 250 nm; thus 2P and 3P absorptions are expected at ~ 500 and ~700 nm respectively. After optical excitation, a serotonin molecule relaxes vibrationally through environmental interactions and releases a fluorescent photon with an energy less than that of the total energy absorbed (~340 nm). In these imaging studies we utilize low energy red photons (740 nm) and a three photon mechanism to activate native serotonin fluorescence. Red light 3P excitation alleviates the need for deep UV optics and their associated aberrations and low transmissivities. Nonlinear excitation is also intrinsically localized to the focal plane and thus is potentially less invasive than single-photon techniques. We have previously shown that 3P microscopy of serotonin-granules in RBL-2H3 cells is possible (Maiti 1997). Here we report 3P imaging measurements of those cells undergoing antigen stimulated secretion. Sampling at one frame every 10-20 seconds, we observe each vesicle's contents to disappear suddenly with no evidence for granule motion. Single granule release rates can be determined and indicate release that is spatially coupled and sometimes oscillatory. Upon exodus granules often leave behind clearly defined footprints, suggesting undissolved serotonin still bound to the granular matrix. Denk, W., D. W. Piston and W. W. Webb (1995). Two-photon molecular excitation in laser-scanning microscopy. Handbook of Biological Confocal Microscopy. J. B. Pawley. New York, Plenum Press: 445-458. Denk, W., J. H. Strickler and W. W. Webb (1990). "Two-photon laser scanning Maiti, S., J. B. Shear, R. M. Williams, W. R. Zipfel and W. W. Webb (1997). "Measuring serotonin distribution in live cells with three-photon excitation" Science 275:530-532. Williams, R. M., D. W. Piston and W. W. Webb (1994). "Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry." FASEB J. 8: 804-813. Xu, C. and W. W. Webb (1996). "Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm." J. Opt. Soc. Am. B 13(3): 481-491. Xu, C., et al. (1996). "Multiphoton fluorescence excitaiton: new spectral windows for biological nonlinear microscopy." Proc. Natl. Acad. Sci. 93: 10763-10768.